Genome-wide target analysis of Shh-activated transcription network in limb bud

我们的长期目标是解开将基因调控和表达的早期模式与结构的最终实现联系起来的步骤，以作为信号网络如何协调复杂组织形成的范例。为了实现这一目标，我们正在开发几种结合的遗传学、基因组和蛋白质组学方法来研究肢体发育过程中的转录因子和调节级联反应，最终目的是阐明在早期诱导前后方(AP)模式和不同手指(具有不同数量、长度和形状的指骨)的形态发生之间的调控等级。了解转录因子如何在正常发育过程中协调生长和形态发生，将促进我们对如何治疗遗传疾病和癌症的理解，这些疾病和癌症是由于这些调控成分发生突变或异常表达而引起的。Hoxd基因如何指示手指识别的特征(如关节数量、形状、大小)，Hoxd和Gli3靶标之间有什么关系？了解这种三维结构是如何形成的，对于理解器官发生是如何实现的通常是相关的。最近报道了一些5Hoxd体内靶点(Shh增强子，Hand2启动子)，尽管还不是很好地描述。定义5Hoxd函数的时间窗口在芯片分析的肢体阶段的选择中将是重要的。我们的遗传学结果检测了不同时间移除Hoxd基因功能的效果，表明这些基因在手指发育的相对较晚阶段调节手指形态和关节形成，当时手指的前驱射线已经开始出现。我们已经为CHIP开发了几种抗体，并与Steve Vokes(德克萨斯大学奥斯汀分校)合作，成功地为具有抗标签的CHIP设计了一个表位标记的Hoxd13条件转基因等位基因。这种可诱导的转基因还将有助于靶标的生物验证，以及我们在实验室中已经拥有的条件敲除等位基因。结果将与沃克斯博士实验室的预期结果相关联，沃克斯博士也在分析Gli3靶点，他已经为我们提供了一种表位标记的Gli3表达小鼠品系。识别Hoxd和Gli3靶点将有助于深入了解共调控基因和Gli3-Hoxd的作用，并阐明Hoxd基因在肢体形态发生中的后期效应。还将分析由Hoxd和Gli3在肢体中调控的转录网络与Shh途径靶标的关系，Shh途径靶标形成两个不同的类别，需要瞬时或持续的信号才能稳定激活。通过这种方式，我们希望揭示导致形成具有不同数量的节段和关节的已定义手指形态的调节级联。Gli和Hox基因在一些癌症中也异常共表达，可能参与了它们的发病机制，这些研究也将阐明它们在这些背景下可能扮演的角色。

Our long term goal is to unravel the steps linking early patterns of gene regulation and expression with the ultimate realization of structure to serve as a paradigm for how signaling networks orchestrate the formation of a complex tissue. To accomplish this, we are developing several combined genetic and genomic, and proteomic approaches to study transcription factors and regulatory cascades operating during limb development with the ultimate aim of elucidating the regulatory hierarchy between early induction of antero-posterior (AP) pattern and the morphogenesis of distinct digits (with different numbers, lengths and shapes of phalanges). Learning how transcription factors orchestrate growth and morphogenesis during normal development will advance our understanding of how to treat genetic diseases and cancers that arise when such regulatory components are either mutated or expressed abnormally. How do Hoxd genes instruct features of digit identity (such as numbers of joints, shape, size) and what is the relation between Hoxd and Gli3 targets? Learning how this 3-dimensional structure forms will be generally relevant for understanding how organogenesis is achieved. A few 5Hoxd in vivo targets have been reported recently (Shh enhancer, Hand2 promoter), albeit not well-characterized. Defining time windows for 5Hoxd functions will be important in choice of limb stages for ChIP analysis. Our genetic results examining effects of removing Hoxd gene function at different times indicates that these genes regulate digit morphology and joint formation at relatively late stages of digit development, when precursor rays for digits have begun to appear. We have developed several antibodies for ChIP, and we have successfully engineered an epitope-tagged Hoxd13 conditional transgene allele for ChIP with anti-tag in collaboration with Steve Vokes (U. Texas, Austin). This inducible transgene will also facilitate biological validation of targets, along with a conditonal knock-out allele that we already have in the lab. Results will be correlated together with anticipated results from Dr. Vokes lab, who is also analyzing Gli3 targets, and who has made an epitope-tagged Gli3 expressing mouse line available to us. Identifying Hoxd and Gli3 targets will provide insight into co-regulated genes and Gli3-Hoxd roles as well as illuminating late effectors of Hoxd genes in limb morphogenesis. The transcriptional network regulated by Hoxd and Gli3 in the limb will also be analyzed in relation to Shh-pathway targets that form two distinct classes, requiring either transient or sustained signaling for their stable activation. In this manner, we hope to uncover the regulatory cascade leading to formation of defined digit morphologies with distinct numbers of segments and joints. Gli and Hox genes are also aberrantly co-expressed in some cancers and may contribute to their pathogenesis, and these studies will also shed light on their possible roles in these contexts.